(80) Profiling the Direct and Indirect Contributions of VGLL1 to the Human Trophectoderm.

Ruben I. Calderon¹, Walee B. Shaik², Claire Zheng¹, Jennifer N. Chousal², Francesca Soncin².

1. University of California San Diego, School of Biological Sciences. La Jolla, CA. United States of America.

2. University of California San Diego, Department of Pathology. La Jolla, CA. United States of America.

Abstract Text:

The trophectoderm (TE) plays a critical role in the establishment of a viable pregnancy by contributing to blastocyst hatching, attachment, implantation, and initiation of the maternal/fetal interface. The spatio-temporal regulation of signaling pathways and transcriptional networks underlying normal and abnormal TE/trophoblast development in humans remain poorly understood yet contribute to a significant loss of pregnancies and placenta dysfunctions that impact pregnancy outcomes. We first reported Vestigial-like Family Member 1 (VGLL1), a coactivator of TEAD transcription factors, as a human placenta-specific marker. In human blastocysts, VGLL1 expression is restricted to the TE, where it co-localizes with TEAD4 and TEAD1. We have established a protocol for in vitro derivation of human TE-like and trophoblast stem cells (hTSC) from human pluripotent stem cells (hPSC). Interestingly, hPSC with constitutive VGLL1 knockdown fail to differentiate into TE-like cells and subsequently do not adapt to hTSC culture conditions, indicated by significantly decreased expression of key TE/trophoblast lineage regulators, including GATA3. Therefore, we further investigated the direct and indirect contributions of VGLL1 to human TE specification.
    We generated three distinct hPSC lines that express a doxycycline (DOX) inducible dCas9-VPR CRISPR activation system and gRNA for VGLL1 (iVGLL1a), to test if ectopic expression of VGLL1 was sufficient to direct TE/trophoblast identity. Though VGLL1 expression alone was insufficient to establish bona fide TSCs, we observed up-regulation of TE/trophoblast genes, including GATA3, ENPEP, and EGFR by qPCR, flow cytometry, and RNA-sequencing (RNA-seq) analysis (adj. P < 0.05). Gene ontology analysis suggested activation of the WNT/b-catenin signaling pathway in response to ectopic VGLL1 expression, including WNT signaling components, CTNNB1, FZD2, LEF1, LGR4, and LRP4/5/6. Additionally, we observed up-regulation of WNT-dependent mesoderm markers, EOMES, TBXT, and MIXL1 in DOX-treated iVGLL1a (adj. P < 0.05). Inhibition of WNT signaling with the small molecule IWP2 abrogated GFP expression of a TCF/LEF-dependent reporter and mesodermal gene expression, but enhanced expression of TE/trophoblast lineage markers GATA3 and EGFR.
     We then performed chromatin immunoprecipitation sequencing (ChIP-seq) using VGLL1 and TEAD4 antibodies to assess genome wide occupancy and determine genes directly regulated by VGLL1. We identified 7241 peaks occupied by VGLL1, localized primarily at promoters (20%), introns (44.4%), and intergenic regions (30.6%) at 5039 genes specifically enriched in human embryo trophectoderm (adj. P < 0.0001), including GATA3, ENPEP, and EGFR. We did not detect VGLL1 occupancy at mesoderm lineage genes EOMES, MIXL1, or TBXT, though there were peaks at WNT/b-catenin pathway components including FZD2, LGR4, and LRP4/5/6. Interestingly, our integrated data analyses indicate that VGLL1 directly regulates the expression of lysine demethylase KDM6B, which in murine in vivo models, is required for blastocyst implantation and expression of key TE-regulators Gata3 and Cdx2. In humans, VGLL1 and KDM6B are more highly expressed in the polar TE, which attaches to the uterine wall during implantation. It remains unclear whether KDM6B is involved in human implantation, but our results show that chemical inhibition of KDM6B demethylase activity phenocopies loss of VGLL1 and results in the blunted expression of GATA3 and CDX2, in vitro. Taken together, our data suggest that VGLL1 is required for the activation of WNT/b-catenin signaling pathway, is important for proper hTSC gene expression, and enables epigenetic changes to TE/trophoblast lineage-specific genes, thus contributes both directly and indirectly to establish the TE/trophoblast identity.